The extent to which cancer tissue is completely removed during primary surgery is a critical prognostic indicator of local recurrence and overall patient survival. However, current techniques deployed to identify and remove cancer cells during surgery are terribly inadequate, often relying on a combination of visual inspection, palpation, and co-registered pre-operative imaging. Incomplete resection during breast conserving surgery has emerged as a particularly prevalent problem, with some studies reporting repeat surgery rates over 50%, representing an enormous mental and physical cost for patients and the health care system. This project aims to dramatically reduce this rate by advancing a novel optical imaging strategy to rapidly identify incomplete tumor resection during surgery. Significant effort has been directed towards developing fluorescent imaging agents targeted to abnormal molecular features characteristic of tumor cells, such as receptor expression. However, administering these agents in humans is a significant regulatory challenge. Topical application of targeted probes to excised fresh tissue during surgery is an attractive alternative which precludes administering diagnostic contrast agents to humans. While conceptually simple, the practical challenges involved in implementing this approach, especially the prevalence of non-specific uptake in normal tissues, constitute a nearly intractable problem. To address this problem, we have developed a dual-probe difference specimen imaging (DDSI) strategy which deploys a non-targeted counterpart probe to account for non-specific uptake in normal tissue. Applying this technique in breast tumor models increased tumor-to-normal contrast by between 5 and 340-fold, depending on the normal tissue type considered, over approaches which use a single targeted probe. These extremely promising results motivate this project, dedicated to advancing and validating DDSI on multiple fronts. Specifically, we will develop and evaluate a multi-channel wide-field fluorescence imaging system dedicated to biomarker-specific DDSI in the operating room (OR). This system will incorporate depth-sensitive capabilities and a novel diagnostic projection technology for intuitive integration in th OR. We will also apply small-molecule chemical synthesis to develop and test novel fluorescent probes with properties favorable to DDSI. We will deploy these technologies to establish the diagnostic performance of DDSI against a single breast cancer biomarker, and show that DDSI against multiple biomarkers simultaneously, i.e. truly personalized imaging based on pre-surgical biopsy information, provides even higher diagnostic performance. Finally, we will complete a clinical pilot study to evaluate the feasibility of clinical translation and confirm tha multi-target DDSI provides higher tumor contrast than single-target DDSI in lumpectomy specimens. This clinical pilot study will establish the groundwork for a future clinical trial.